Peppered with lead: An environmental forensics approach to identify the source of rising blood lead levels.

Despite the phase-out of lead-based products, lead contamination can still present a contemporary risk to public health. In situations where elevated blood lead cannot be attributed to common sources, detailed environmental investigation is needed to identify more elusive sources and manage harmful exposure pathways. We apply a forensics approach to assess common and elusive sources of lead in the home environment of two individuals with fluctuating blood lead levels in Sydney, Australia. Using multiple analytical lines of evidence (portable X-Ray Fluorescence spectrometry (pXRF), inductively coupled-plasma mass spectrometry (ICP-MS), lead isotopic compositional analysis (PbIC) and haematological assessment) a pewter pepper grinder containing lead (>6000 mg/kg; 70% bioavailable) was identified as a potential source. After removing the pepper grinder from the home, the couple's blood lead decreased to below the Australian intervention level of 5 µg/dL within a year (Person A: from 12.5 µg/dL in August 2020 to 4.4 µg/dL in March 2022; and Person B: 15.4 µg/dL in August 2020 to 2.1 µg/dL in July 2021). This case study demonstrates how environmental science investigations can play a crucial role in supporting people to take evidence-based action to improve their health.

Radiocarbon determination of fossil and contemporary carbon contribution to aerosol in the Pacific Islands.

Combustion emissions are of growing concern across all Pacific Island Countries, which account for >10,000 km2 of the earth's surface area; as for many other small island states globally. Apportioning emissions inputs for Suva, the largest Pacific Island city, will aid in development of emission reduction strategies. Total suspended particulate (TSP) and fine particulate (PM2.5) samples were collected for Suva City, a residential area (Kinoya, TSP) and a mainly ocean-influenced site (Suva Point, TSP) from 2014 to 2015. Percentages of contemporary and fossil carbon were determined by radiocarbon analysis (accelerator mass spectrometry); for non­carbonate carbon (NCC), elemental carbon (EC) and organic carbon (OC). Source contributions to particulate matter were identified and the accuracy of previous emissions inventory and source apportionment studies was evaluated. Suva Point NCC concentrations (2.7 ±â€¯0.4 µg/m3) were four times lower than for City (13 ±â€¯2 µg/m3 in TSP) and Kinoya (13 ±â€¯1 µg/m3 in TSP); demonstrating the contribution of land-based emissions activities in city and residential areas. In Suva City, total NCC in air was 81% (79%-83%) fossil carbon, from vehicles, shipping, power generation and industry; whilst in the residential area, 48% (46%-50%) of total NCC was contemporary carbon; reflecting the higher incidence of biomass and waste burning and of cooking activities. Secondary organic fossil carbon sources contributed >36% of NCC mass at the city and >29% at Kinoya; with biogenic carbon being Kinoya's most significant source (approx. 30% of NCC mass). These results support the previous source apportionment studies for the city area; yet show that, in line with emissions inventory studies, biomass combustion contributes more PM2.5 mass in residential areas. Hence air quality management strategies need to target open burning activities as well as fossil fuel combustion.

Reducing mortality risk by targeting specific air pollution sources: Suva, Fiji.

Health implications of air pollution vary dependent upon pollutant sources. This work determines the value, in terms of reduced mortality, of reducing ambient particulate matter (PM2.5: effective aerodynamic diameter 2.5µm or less) concentration due to different emission sources. Suva, a Pacific Island city with substantial input from combustion sources, is used as a case-study. Elemental concentration was determined, by ion beam analysis, for PM2.5 samples from Suva, spanning one year. Sources of PM2.5 have been quantified by positive matrix factorisation. A review of recent literature has been carried out to delineate the mortality risk associated with these sources. Risk factors have then been applied for Suva, to calculate the possible mortality reduction that may be achieved through reduction in pollutant levels. Higher risk ratios for black carbon and sulphur resulted in mortality predictions for PM2.5 from fossil fuel combustion, road vehicle emissions and waste burning that surpass predictions for these sources based on health risk of PM2.5 mass alone. Predicted mortality for Suva from fossil fuel smoke exceeds the national toll from road accidents in Fiji. The greatest benefit for Suva, in terms of reduced mortality, is likely to be accomplished by reducing emissions from fossil fuel combustion (diesel), vehicles and waste burning.

Airborne ultrafine particles in a Pacific Island country: Characteristics, sources and implications for human exposure.

The Pacific Islands carry a perception of having clean air, yet emissions from transport and burning activities are of concern in regard to air quality and health. Ultrafine particle number concentrations (PNCs), one of the best metrics to demonstrate combustion emissions, have not been measured either in Suva or elsewhere in the Islands. This work provides insight into PNC variation across Suva and its relationship with particle mass (PM) concentration and composition. Measurements over a short monitoring campaign provide a vignette of conditions in Suva. Ambient PNCs were monitored for 8 day at a fixed location, and mobile PNC sampling for two days. These were compared with PM concentration (TSP, PM10, PM2.5, PM1) and are discussed in relation to black carbon (BC) content and PM2.5 sources, determined from elemental concentrations; for the October 2015 period and longer-term data. Whilst Suva City PM levels remained fairly low, PM2.5 = 10-12 µg m-3, mean PNC (1.64 ± 0.02 × 104 cm-3) was high compared to global data. PNCs were greater during mobile sampling, with means of 10.3 ± 1.4 × 104 cm-3 and 3.51 ± 0.07 × 104 cm-3 when travelling by bus and taxi, respectively. Emissions from road vehicles, shipping, diesel and open burning were identified as PM sources for the October 2015 period. Transport related ultrafine particle emissions had a significant impact on microscale ambient concentrations, with PNCs near roads being 1.5 to 2 times higher than nearby outdoor locations and peak PNCs occurring during peak traffic times. Further data, particularly on transport and wet-season exposures, are required to confirm results. Understanding PNC in Suva will assist in formulating effective air emissions control strategies, potentially reducing population exposure across the Islands and in developing countries with similar emission characteristics. Suva's PNC was high in comparison to global data; high exposures were related to transport and combustion emissions, which were also identified as significant PM2.5 sources.

Chemical, biological, and DNA markers for tracing slaughterhouse effluent.

Agricultural practices, if not managed correctly, can have a negative impact on receiving environments via waste disposal and discharge. In this study, a chicken slaughter facility on the rural outskirts of Sydney, Australia, has been identified as a possible source of persistent effluent discharge into a peri-urban catchment. Questions surrounding the facility's environmental management practices go back more than four decades. Despite there having never been a definitive determination of the facility's impact on local stream water quality, the New South Wales Environment Protection Authority (NSW EPA) has implemented numerous pollution reduction requirements to manage noise and water pollution at the slaughter facility. However, assessment of compliance remains complicated by potential additional sources of pollution in the catchment. To unravel this long-standing conundrum related to water pollution we apply a forensic, multiple lines of evidence approach to delineate the origin of the likely pollution source(s). Water samples collected between 2014 and 2016 from irrigation pipes and a watercourse exiting the slaughter facility had elevated concentrations of ammonia (max: 63,000µg/L), nitrogen (max: 67,000µg/L) and phosphorus (max: 39,000µg/L), which were significantly higher than samples from adjacent streams that did not receive direct runoff from the facility. Arsenic, sometimes utilised in growth promoting compounds, was detected in water discharging from the facility up to ~4 times (max 3.84µg/L) local background values (<0.5µg/L), with inorganic As(∑V+III) being the dominant species. The spatial association of elevated water pollution to the facility could not unequivocally distinguish a source and consequently DNA analysis of a suspected pollution discharge event was undertaken. Analysis of catchment runoff from several local streams showed that only water sampled at the downstream boundary of the facility tested positive for chicken DNA, with traces of duck DNA being absent, which was a potential confounder given that wild ducks are present in the area. Further, PCR analysis showed that only the discharge water emanating from the slaughter facility tested positive for a generalized marker of anthropogenic pollution, the clinical class 1 integron-integrase gene. The environmental data collected over a three-year period demonstrates that the slaughter facility is indisputably the primary source of water-borne pollution in the catchment. Moreover, application of DNA and PCR for confirming pollution sources demonstrates its potential for application by regulators in fingerprinting pollution sources.

Geochemical sources, forms and phases of soil contamination in an industrial city.

This study examines current soil contamination in an Australian industrial city, Newcastle. Public (roadside verges and parks) and private (homes) surface soils (n=170) contained metal(loid)s elevated above their respective Australian Health Investigation Levels (HIL). Lead (Pb), the most common contaminant in the city, exceeds the HIL for residential soils (HIL-A, 300mg/kg) in 88% of private soils (median: 1140mg/kg). In-vitro Pb bio-accessibility analysis of selected soils (n=11) using simulated gastric fluid showed a high affinity for Pb solubilisation (maximum Pb concentration: 5190mg/kg, equating to 45% Pb bio-accessibility). Highly soluble Pb-laden Fe- and Mn-oxides likely contribute to the bio-accessibility of the Pb. Public and private space surface soils contain substantially less radiogenic Pb (range: 208Pb/207Pb: 2.345-2.411, 206Pb/207Pb: 1.068-1.312) than local background soil (208Pb/207Pb: 2.489, 206Pb/207Pb: 1.198), indicating anthropogenic contamination from the less radiogenic Broken Hill type Pb ores (208Pb/207Pb: 2.319, 206Pb/207Pb: 1.044). Source apportionment using Pb isotopic ratio quantification and soil mineralogy indicate the city's historic copper and steel industries contributed the majority of the soil contaminants through atmospheric deposition and use of slag waste as fill material. High-temperature silicates and oxides combined with rounded particles in the soil are characteristic of smelter dust emissions. Additionally, a preliminary investigation of polycyclic aromatic hydrocarbons in soils, sometimes associated with ferrous metal smelting, coal processing or burning of fossil fuels, shows that these too pose a health exposure risk (calculated in comparison to benzo(a)pyrene: n=12, max: 13.5mg/kg, HIL: 3mg/kg).

Widespread copper and lead contamination of household drinking water, New South Wales, Australia.

This study examines arsenic, copper, lead and manganese drinking water contamination at the domestic consumer's kitchen tap in homes of New South Wales, Australia. Analysis of 212 first draw drinking water samples shows that almost 100% and 56% of samples contain detectable concentrations of copper and lead, respectively. Of these detectable concentrations, copper exceeds Australian Drinking Water Guidelines (ADWG) in 5% of samples and lead in 8%. By contrast, no samples contained arsenic and manganese water concentrations in excess of the ADWG. Analysis of household plumbing fittings (taps and connecting pipework) show that these are a significant source of drinking water lead contamination. Water lead concentrations derived for plumbing components range from 108µg/L to 1440µg/L (n=28, mean - 328µg/L, median - 225µg/L). Analysis of kitchen tap fittings demonstrates these are a primary source of drinking water lead contamination (n=9, mean - 63.4µg/L, median - 59.0µg/L). The results of this study demonstrate that along with other potential sources of contamination in households, plumbing products that contain detectable lead up to 2.84% are contributing to contamination of household drinking water. Given that both copper and lead are known to cause significant health detriments, products for use in contact with drinking water should be manufactured free from copper and lead.

gD-Independent Superinfection Exclusion of Alphaherpesviruses.

UNLABELLED: Many viruses have the capacity to prevent a cell from being infected by a second virus, often termed superinfection exclusion. Alphaherpesviruses, including the human pathogen herpes simplex virus 1 (HSV-1) and the animal herpesvirus pseudorabies virus (PRV), encode a membrane-bound glycoprotein, gD, that can interfere with subsequent virion entry. We sought to characterize the timing and mechanism of superinfection exclusion during HSV-1 and PRV infection. To this end, we utilized recombinant viruses expressing fluorescent protein (FP) markers of infection that allowed the visualization of viral infections by microscopy and flow cytometry as well as the differentiation of viral progeny. Our results demonstrated the majority of HSV-1- and PRV-infected cells establish superinfection exclusion by 2 h postinfection. The modification of viral infections by virion inactivation and phosphonoacetic acid, cycloheximide, and actinomycin D treatments indicated new protein synthesis is needed to establish superinfection exclusion. Primary infection with gene deletion PRV recombinants identified that new gD expression is not required to establish superinfection exclusion of a secondary viral inoculum. We also identified the timing of coinfection events during axon-to-cell spread, with most occurring within a 2-h window, suggesting a role for cellular superinfection exclusion during neuroinvasive spread of infection. In summary, we have characterized a gD-independent mechanism of superinfection exclusion established by two members of the alphaherpesvirus family and identified a potential role of exclusion during the pathogenic spread of infection. IMPORTANCE: Superinfection exclusion is a widely observed phenomenon initiated by a primary viral infection to prevent further viruses from infecting the same cell. The capacity for alphaherpesviruses to infect the same cell impacts rates of interviral recombination and disease. Interviral recombination allows genome diversification, facilitating the development of resistance to antiviral therapeutics and evasion of vaccine-mediated immune responses. Our results demonstrate superinfection exclusion occurs early, through a gD-independent process, and is important in the directed spread of infection. Identifying when and where in an infected host viral genomes are more likely to coinfect the same cell and generate viral recombinants will enhance the development of effective antiviral therapies and interventions.

Evaluation and assessment of the efficacy of an abatement strategy in a former lead smelter community, Boolaroo, Australia.

This study examines the recent soil Lead Abatement Strategy (LAS) in Boolaroo, New South Wales, Australia, that was designed to "achieve a reduction in human exposure to lead dust contamination in surface soils". The abatement programme addressed legacy contamination of residential areas following closure of lead smelting operations in 2003 at the Pasminco Cockle Creek Smelter (PCCS). The principal objective of the LAS was to "cap and cover" lead-contaminated soils within the urban environment surrounding the PCCS. Soil lead concentrations of 2500-5000 mg/kg were scheduled for removal and replacement, while concentrations between 1500 and 2500 mg/kg were replaced only under limited circumstances. To date, there has been no industry, government or independent assessment of the clean-up programme that involved >2000 homes in the township of Boolaroo. Thus, by measuring post-abatement soil lead concentrations in Boolaroo, this study addresses this knowledge gap and evaluates the effectiveness of the LAS for reducing the potential for lead exposure. Soil lead concentrations above the Australian residential soil health investigation level value for residential soils (300 mg/kg) were identified at all but one of the residential properties examined (n = 19). Vacuum dust samples (n = 17) from the same homes had a mean lead concentration of 495 mg/kg (median 380 mg/kg). Bio-accessibility testing revealed that lead in household vacuum dust was readily accessible (% bio-accessible) (mean = 92 %, median = 90 %), demonstrating that the risk of exposure via this pathway remains. Assessment of a limited number of properties (n = 8) where pre-abatement soil lead levels were available for comparison showed they were not statistically different to post-abatement. Although the LAS did not include treatment of non-residential properties, sampling of community areas including public sports fields, playgrounds and schools (n = 32) was undertaken to determine the contamination legacy in these areas. Elevated mean soil lead concentrations were found across public lands: sports fields = 5130 mg/kg (median = 1275 mg/kg), playgrounds and schools = 812 mg/kg (median = 920 mg/kg) and open space = 778 mg/kg (median = 620 mg/kg). Overall, the study results show that the LAS programme that was dominated by a "cap and cover" approach to address widespread lead contamination was inadequate for mitigating current and future risk of lead exposures.

Identification of the sources of metal (lead) contamination in drinking waters in north-eastern Tasmania using lead isotopic compositions.

This study utilises a range of scientific approaches, including lead isotopic compositions, to differentiate unknown sources of ongoing lead contamination of a drinking water supply in north-eastern Tasmania, Australia. Drinking water lead concentrations are elevated above the Australian Drinking Water Guideline (10 µg/L), reaching 540 µg/L in the supply network. Water lead isotopic compositions from the town of Pioneer ((208)Pb/(207)Pb 2.406, (206)Pb/(207)Pb 1.144 to (208)Pb/(207)Pb 2.360, (206)Pb/(207)Pb 1.094) and Ringarooma ((208)Pb/(207)Pb 2.398, (206)Pb/(207)Pb 1.117) are markedly different from the local bedrock ((208)Pb/(207)Pb 2.496, (206)Pb/(207)Pb 1.237). The data show that the lead in the local waters is sourced from a combination of dilapidated drinking water infrastructure, including lead jointed pipelines, end-of-life polyvinyl chloride pipes and household plumbing. Drinking water is being inadvertently contaminated by aging infrastructure, and it is an issue that warrants investigation to limit the burden of disease from lead exposure.

Us9-Independent Axonal Sorting and Transport of the Pseudorabies Virus Glycoprotein gM.

Axonal sorting and transport of fully assembled pseudorabies virus (PRV) virions is dependent on the viral protein Us9. Here we identify a Us9-independent mechanism for axonal localization of viral glycoprotein M (gM). We detected gM-mCherry assemblies transporting in the anterograde direction in axons. Furthermore, unlabeled gM, but not glycoprotein B, was detected by Western blotting in isolated axons during Us9-null PRV infection. These results suggest that gM differs from other viral proteins regarding axonal transport properties.

Lead and zinc dust depositions from ore trains characterised using lead isotopic compositions.

This study investigates an unusual source of environmental lead contamination - the emission and deposition of lead and zinc concentrates along train lines into and out of Australia's oldest silver-lead-zinc mine at Broken Hill, Australia. Transport of lead and zinc ore concentrates from the Broken Hill mines has occurred for more than 125 years, during which time the majority was moved in uncovered rail wagons. A significant amount of ore was lost to the adjoining environments, resulting in soil immediately adjacent to train lines elevated with concentrations of lead (695 mg kg(-1)) and zinc (2230 mg kg(-1)). Concentrations of lead and zinc decreased away from the train line and also with depth shown in soil profiles. Lead isotopic compositions demonstrated the soil lead contained Broken Hill ore in increasing percentages closer to the train line, with up to 97% apportioned to the mined Broken Hill ore body. SEM examination showed ceiling dusts collected from houses along the train line were composed of unweathered galena particles, characteristic of the concentrate transported in the rail wagons. The loss of ore from the uncovered wagons has significantly extended the environmental footprint of contamination from local mining operations over an area extending hundreds of kilometres along each of the three train lines.

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius.

This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 µM at pH 5), high catalytic efficiency (4.75 × 10(5) M(-1) s(-1) at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35 ± 0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.

Identification of lead sources in residential environments: Sydney Australia.

Interior and exterior dust, soil and paint were analysed at five brick urban Sydney homes over 15 months to evaluate temporal variations and discriminate sources of lead (Pb) exposure. Exterior dust gauge Pb loading rates (µg/m(2)/28 days), interior vacuum dust Pb concentrations (mg/kg) and interior petri-dish Pb loading rates (µg/m(2)/28 days), were correlated positively with soil Pb concentrations. Exterior dust gauge Pb loading rates and interior vacuum dust Pb concentrations peaked in the summer. Lead isotope and Pb speciation (XAS) were analysed in soil and vacuum dust samples from three of the five houses that had elevated Pb concentrations. Results show that the source of interior dust lead was primarily from soil in two of the three houses and from soil and Pb paint in the third home. IEUBK child blood Pb modelling predicts that children's blood Pb levels could exceed 5 µg/dL in two of the five houses.

Identification of environmental lead sources and pathways in a mining and smelting town: Mount Isa, Australia.

Lead (Pb) concentrations and isotopic compositions from soils, dusts and aerosols from public land and residential lots adjacent to the copper and Pb mine and smelter at Mount Isa, Australia, were examined to understand the sources and risks of environmental Pb exposure. Urban soil samples contain elevated Pb concentrations (mean 1560 mg/kg), of which 45-85% of the Pb is bioaccessible. The Pb isotopic composition of surface soils (0-2 cm), aerosols and dusts ((206)Pb/(207)Pb, (208)Pb/(207)Pb range: 1.049, 2.322-1.069, 2.345) are dominated by Pb derived from the Mount Isa Pb-zinc ore bodies. Underlying soil horizons (10-20 cm) have distinctly different Pb isotopic compositions ((206)Pb/(207)Pb, (208)Pb/(207)Pb range: 1.093, 2.354-1.212, 2.495). Surface soil-, dust- and aerosol-Pb are derived predominantly from smelter emissions and fugitive mining sources and not from in situ weathered bedrock. Remediation strategies should target legacy and ongoing sources of environmental Pb to mitigate the problem of Pb exposure at Mount Isa.

Making the case: married versus separate models of alphaherpes virus anterograde transport in axons.

Alphaherpesvirus virions infect neurons and are transported in axons for long distance spread within the host nervous system. The assembly state of newly made herpesvirus particles during anterograde transport in axons is an essential question in alphaherpesvirus biology. The structure of the particle has remained both elusive and controversial for the past two decades, with conflicting evidence from EM, immunofluorescence, and live cell imaging studies. Two opposing models have been proposed-the Married and Separate Models. Under the Married Model, infectious virions are assembled in the neuronal cell body before sorting into axons and then traffic inside a transport vesicle. Conversely, the Separate Model postulates that vesicles containing viral membrane proteins are sorted into axons independent of capsids, with final assembly of mature virions occurring at a distant egress site. Recently, a complementary series of studies employing high-resolution EM and live cell fluorescence microscopy have provided evidence consistent with the Married Model, whereas other studies offer evidence supporting the Separate Model. In this review, we compare and discuss the published data and attempt to reconcile divergent findings and interpretations as they relate to these models.

Visualization of an alphaherpesvirus membrane protein that is essential for anterograde axonal spread of infection in neurons.

UNLABELLED: Pseudorabies virus (PRV), an alphaherpesvirus with a broad host range, replicates and spreads in chains of synaptically connected neurons. The PRV protein Us9 is a small membrane protein that is highly conserved among alphaherpesviruses and is essential for anterograde axonal spread in neurons. Specifically, the Us9 protein is required for the sorting of newly assembled PRV particles into axons. However, the molecular details underlying the function of Us9 are poorly understood. Here we constructed PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize axonal transport of viral particles in infected rat superior cervical ganglion neurons. We show that GFP-Us9-labeled structures are transported exclusively in the anterograde direction within axons. Additionally, the vast majority of anterograde-directed capsids (labeled with VP26-monomeric red fluorescent protein) and a viral membrane protein (labeled with glycoprotein M fused to mCherry) are cotransported with GFP-Us9 in the anterograde direction. In contrast, during infection with PRV strains that express nonfunctional mutant GFP-Us9 proteins, cotransport of mutant GFP-Us9 with capsids in axons is abolished. These findings show that axonal sorting of progeny viral particles is dependent upon the association of viral structures with membranes that contain functional Us9 proteins. This association is required for anterograde spread of infection in neurons. IMPORTANCE: Alphaherpesviruses, such as pseudorabies virus (PRV), are parasites of the mammalian nervous system. These viruses spread over long distances in chains of synaptically connected neurons. PRV encodes several proteins that mediate directed virion transport and spread of infection. Us9 is a highly conserved viral membrane protein that is essential for anterograde neuronal spread of infection. In the absence of Us9, newly replicated viral particles are assembled in the cell body but are not sorted into or transported within axons. Here, we constructed and characterized novel PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize its localization in living neurons during infection. This enabled us to better understand the function of Us9 in facilitating the spread of infection. We show that all viral particles moving in the anterograde direction are labeled with GFP-Us9, suggesting that the presence of Us9 determines the capacity for directed transport within axons.

Herpesvirus replication compartments originate with single incoming viral genomes.

Previously we described a method to estimate the average number of virus genomes expressed in an infected cell. By analyzing the color spectrum of cells infected with a mixture of isogenic pseudorabies virus (PRV) recombinants expressing three fluorophores, we estimated that fewer than seven incoming genomes are expressed, replicated, and packaged into progeny per cell. In this report, we expand this work and describe experiments demonstrating the generality of the method, as well as providing more insight into herpesvirus replication. We used three isogenic PRV recombinants, each expressing a fluorescently tagged VP26 fusion protein (VP26 is a capsid protein) under the viral VP26 late promoter. We calculated a similar finite limit on the number of expressed viral genomes, indicating that this method is independent of the promoter used to transcribe the fluorophore genes, the time of expression of the fluorophore (early versus late), and the insertion site of the fluorophore gene in the PRV genome (UL versus US). Importantly, these VP26 fusion proteins are distributed equally in punctate virion assembly structures in each nucleus, which improves the signal-to-noise ratio when determining the color spectrum of each cell. To understand how the small number of genomes are distributed among the replication compartments, we used a two-color fluorescent in situ hybridization assay. Most viral replication compartments in the nucleus occupy unique nuclear territories, implying that they arose from single genomes. Our experiments suggest a correlation between the small number of expressed viral genomes and the limited number of replication compartments.